Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through

Pregnancy enhances the innate immune response
in experimental cutaneous leishmaniasis through
hormone-modulated nitric oxide production
Yaneth Osorio,*,†,‡,1 Diana L. Bonilla,† Alex G. Peniche,† Peter C. Melby,*,‡
and Bruno L. Travi*,†,‡,1,2
*Department of Medicine, University of Texas, Health Science Center, San Antonio, Texas, USA; †Centro
Internacional de Entrenamiento e Investigaciones Medicas-CIDEIM, Cali, Colombia; and ‡Research Service,
Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, San Antonio, Texas, USA
Abstract: The maintenance of host defense during pregnancy may depend on heightened innate
immunity. We evaluated the immune response
of pregnant hamsters during early infection
with Leishmania (Viannia) panamensis, a cause
of American cutaneous leishmaniasis. At 7 days
post-infection, pregnant animals showed a lower
parasite burden compared with nonpregnant
controls at the cutaneous infection site (Pⴝ
0.0098) and draining lymph node (Pⴝ0.02).
Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (Pⴝ0.018 each). This enhanced
resistance during pregnancy was associated with
increased expression of inducible NO synthase
(iNOS) mRNA in lymph node cells (Pⴝ0.02) and
higher NO production by neutrophils (Pⴝ
0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high
amounts of NO upon estrogen exposure
(Pⴝ0.05), and addition of the iNOS inhibitor
L-N6-(1-iminoethyl) lysine blocked the induction
of NO production (Pⴝ0.02). Infected, nonpregnant females treated with estrogen showed a
higher percentage of cells producing NO at the
infection site than controls (Pⴝ0.001), which
correlated with lower parasite burdens (Pⴝ
0.036). Cultured macrophages or neutrophils
from estrogen-treated hamsters showed significantly increased NO production and Leishmania
killing compared with untreated controls. iNOS
was identified as the likely source of estrogeninduced NO in primed and naı̈ve macrophages,
as increased transcription was evident by realtime PCR. Thus, the innate defense against
Leishmania infection is heightened during pregnancy, at least in part as a result of estrogenmediated up-regulation of iNOS expression and
NO production. J. Leukoc. Biol. 83:
1413–1422; 2008.
Key Words: Leishmania 䡠 hamster 䡠 estrogen
0741-5400/08/0083-1413 © Society for Leukocyte Biology
INTRODUCTION
During pregnancy, the immune response is modulated to avoid
deleterious effects on the developing fetus. It is generally
accepted that gestation is associated with diminished cellular
immunity [1–3] with decreased expression of proinflammatory
cytokines and NK cell activation, responses that could lead to
fetal abortion [4 – 6]. It is believed that the down-regulation of
adaptive immune responses makes pregnant females more
susceptible to a wide variety of pathogens, most notably Plasmodium spp. [3]. This observation extends to laboratory animals infected with Toxoplasma gondii, Listeria monocytogenes,
Mycobacterium leprae, and Leishmania major [7, 8]. Pregnant
mice infected with L. major developed more severe disease
than nonpregnant controls, which was associated with a skewed
Th2 response characteristic of the susceptible phenotype [7].
Also, a few anecdotal observations in humans indicated that
women infected subclinically with Leishmania donovani or
Leishmania infantum developed overt disease during gestation
[9, 10]. This evidence suggests that pregnancy may increase
the risk of developing leishmaniasis; however, there are no
epidemiological data to confirm this suggestion [11].
On the other hand, several studies have suggested that
heightened innate immunity plays a central role in the maintenance of host defense throughout gestation [1, 12]. Pregnant
women have a higher number of activated peripheral blood
monocytes and granulocytes than nonpregnant women [13–15].
Innate immune function in leishmaniasis is of particular importance, as the effector cells, i.e., macrophages and to some
extent, neutrophils, serve as host cells for the parasite [16, 17].
However, to our knowledge, there are no studies that address
the role of these cells in innate host defense against Leishmania infection in pregnant humans or experimental animals.
1
Current address: Department of Medicine, University of Texas, Health
Science Center, San Antonio, TX, USA.
2
Correspondence: Department of Medicine, University of Texas, Health
Science Center, 7703 Floyd Curl Dr., Mailcode 7881, San Antonio, TX
78229-3900, USA. E-mail: [email protected]
Received February 26, 2007; revised January 11, 2008; accepted January
22, 2008.
doi: 10.1189/jlb.0207130
Journal of Leukocyte Biology Volume 83, June 2008 1413
In the last decades, an increasing body of evidence has
established the relationship between sex hormones and the
immune system. Previous studies demonstrated that increased
concentrations of estrogen led to enhanced macrophage phagocytic activity [18] and that estrogen as well as progesterone and
prolactin stimulated cytotoxic mechanisms through the release
of reactive oxygen species [19, 20]. The production of NO,
which is critical to Leishmania containment in the mouse
model [21], is also enhanced by the high physiological levels of
estrogen and progesterone in pregnant females of different
species. In fact, NO is critical for controlling uterine contractility, cervical ripening, and feto-placental blood flow [22–26].
In pregnant rats, progesterone seems to act synergistically with
NO, playing an important role in embryo implantation [27, 28].
Experimental studies in hamsters infected with L. infantum
indicated that females were protected during lactation [29].
Furthermore, female mice or hamsters were shown to be more
resistant to cutaneous leishmaniasis (caused by Leishmania
mexicana and Leishmania (Viannia) panamensis, respectively)
than males, and this difference depended, at least in hamsters,
on the levels of circulating sex hormones [30, 31]. In this study,
we explored the impact of pregnancy and its related hormones
on the innate immune response to experimental cutaneous
infection with L. panamensis. For this purpose, we used an
established hamster model [31, 32] of American cutaneous
leishmaniasis and demonstrated through in vitro and in vivo
experiments that early parasite control in pregnancy was associated with an estrogen-mediated increase in NO production by
macrophages and neutrophils.
MATERIALS AND METHODS
Parasites
A L. panamensis strain (MHOM/COL/84/1099), which is highly pathogenic for
hamsters, was used in all experiments. The strain was episomally transfected
by electroporation with a luciferase (LUC) reporter gene in a pGL2␣-Neomicin-␣ plasmid as described [33]. Dr. Marc Ouellette (University of Laval,
Canada) kindly provided the LUC reporter plasmid. The transfected Leishmania was selected and cultured in complete Schneider’s culture medium supplemented with 10% FCS, 2 mM glutamine, 100 units/mL penicillin, 100
␮g/mL streptomycin, and 0.04 mg/mL G418 neomycin. The virulence and
pathogenicity of the transfected Leishmania were determined in previous
studies [34]. No significant plasmid loss was observed up to 2 months postinfection (p.i.) in the strain used in the present study.
Parasite burden
Parasite burden in the lesions was determined at 7 days p.i. by luminometry
[33] using a modified method as described by Henao et al. [34]. Briefly, the
whole lesion was excised and immediately homogenized by gentle and thorough scraping of the dermis using a scalpel; subsequently, the tissue was lysed
by incubation for 30 min at room temperature with 200 ␮L 5⫻ lysis buffer (125
mM Tris-HCl, pH 7.8, 10 mM DTT, 50% glycerol, and 5% Triton X-100) and
stored at –70°C until processing. After thawing, the lysate was centrifuged at
10,000 rpm, and 20 ␮L of the supernatant was dispensed in an opaque, white
96-well plate. Fresh assay buffer (80 ␮L; 25 mM Tris-HCl buffer, pH 7.8, 2.67
mM MgSO4, 0.1 mM EDTA, 0.53 mM ATP, 33.3 mM DTT, 4.7 ␮M D-luciferin)
was added to the plate immediately before reading in a luminometer (Anthos,
Austria; 12 wells per reading) using an integration time of 20 s at 37°C and the
automatic addition of 100 ␮L assay buffer. The number of parasites was
extrapolated from a linear standard curve generated with LUC-transfected
amastigotes [34].
1414
Journal of Leukocyte Biology Volume 83, June 2008
Infection of animals
Outbred adult (4 months old), female hamsters were used in the studies.
Animals were maintained according to the Guiding Principles for Biomedical
Research Involving Animals (Council for International Organizations of Medical Sciences), the Colombian Law 84 of 1989 of the “Estatuto Nacional de
Protección de los Animales,” and the Resolution #008430 of 1993, which
complements Law 84. These studies were reviewed and approved by the
Institutional Animal Care and Use Committee of the Centro Internacional de
Entrenamiento e Investigaciones Medicas (CIDEIM; Columbia). Pregnant hamsters were obtained after mating with males during two oestral cycles (8 days).
Seven days after mating, female hamsters and nonmated control females were
inoculated intradermally in the snout with 104 wild-type or LUC-transfected L.
panamensis stationary-phase promastigotes suspended in 0.05 mL PBS. Evaluation of the infection during pregnancy was carried out at 7 days p.i. or
post-parturition at 30 days p.i. Animals were killed in a CO2 chamber following
anesthesia with 50 mg/Kg ketamine clorhydrate (Ketamina威, Holliday-Scott
S.A., Argentina) and 0.02 mg/Kg xylazine (Rompun威, Bayer, Colombia). In
animals killed prior to parturition, pregnancy was confirmed by visual inspection of the uterus.
Cytokine and inducible NO synthase (iNOS;
NOS2) mRNA expression in pregnant hamsters
Cytokine and iNOS mRNA expression was determined ex vivo by RT-PCR
using lesion tissue or draining lymph nodes, cultured with leishmanial antigen
(1⫻106 freeze-thaw, inactivated promastigotes) during 20 h at 37°C, 5% CO2,
in RPMI and 5% FCS. Evaluations were made at 7 or 30 days p.i. The primer
sequences were derived from published sequences [35, 36] and validated for
use in RT-PCR previously [31, 32]. In addition, specific primers for TNF-␣
(forward 5⬘-CACAATCCTCTTCTGCCTGC-3⬘ and reverse 5⬘-TGTCTTTGAGAGACATCCCG-3⬘; expected product, 242 bp) were used. RNA extraction and RT were performed in a final volume of 120 ␮L as described by Osorio
et al. [32]. The cDNA (10 ␮L per sample, equivalent to ⬃200 ng reversetranscribed RNA) was denatured at 94°C during 2 min and amplified by PCR
(94°C 15 s, 55°C 30 s, and 72°C 1 min, with a final 3-min extension at 72°C)
for 30 cycles. Less abundant transcripts (IL-4 and IL-12p40) were subjected to
35 amplification cycles. iNOS expression was determined by RT-PCR in lesion
tissue and cells from draining lymph nodes stimulated with Leishmania antigen, using primers specific for hamster iNOS: forward (exon 12) 5⬘-ACCACACAGCCTCCGAGTCC-3⬘ and reverse (exon 13) 5⬘-CTGCCAGATGTGGGTCTTCC-3⬘; expected product, 200 bp. The expression of iNOS and cytokine (IL-4,
TNF-␣, TGF-␤, IFN-␥, IL-10, IL-12p40m) mRNA was analyzed by densitometry using image analysis (Gel Doc威, Bio-Rad, Hercules, CA, USA) and
normalized to the expression of the hypoxanthine phosphoribosyltransferase
gene (HPRT).
Identification of cell populations by
flow cytometry
Lymphocytes, mononuclear cells, and granulocytes were defined by the size
and granularity of cells [forward-scatter/side-scatter (FSC/SSC)] on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). CD4⫹ T
lymphocytes and B lymphocytes from peripheral blood were identified using
FITC-labeled anti-mouse mAb that cross-react with the corresponding hamster
molecules (GK1.5 and 14-4-4S, PharMingen, San Diego, CA, USA, respectively) [37, 38]. Neutrophils were obtained from pooled, EDTA-treated hamster
peripheral blood following a standard precipitation method with Dextran T-500
(Pharmacia, Uppsala, Sweden) [39]. Cells were then sorted by size and granularity (SSC and FSC) on a FACSCalibur flow cytometer (Becton Dickinson).
All neutrophil preparations had ⬎99% purity confirmed by Giemsa stain and
direct microscopy.
Leishmanicidal activity by flow cytometry
To estimate the proportion of cells containing viable parasites, we infect the
cells with promastigotes previously labeled with 1 ␮M CFSE dye (Molecular
Probes, Eugene, OR, USA). After infection (20 min, 1 h, 34°C, 5% CO2),
viable CFSE Leishmania were detected by gating in the cell population,
excluding small FSC/small SSC extracellular parasites, and analyzing the
positive region of the fluorescence 1 (FL1) channel. Flow cytometry analysis
http://www.jleukbio.org
was accomplished using the CellQuest (BD Biosciences, San Jose, CA, USA)
program.
Treatment of hamsters with estrogen
and progesterone
Parasite replication in macrophages
Hamsters were treated intramuscularly with 2 ␮g 17-␤-estradiol (Sigma Chemical Co.) or 3 ␮g of the active progesterone metabolite pregnane (Sigma
Chemical Co.) every 48 h for 14 days. These treatment schedules were shown
previously to increase plasma levels of these hormones [40]. Protocols of
infection and in vitro assays were followed as described for pregnant animals.
Hamsters were infected intradermally in the snout with L. panamensis as
described before on the 7th day of hormone treatment. At 7 days p.i. (14th day
of hormone treatment), the animals were killed, and the parasite burden was
determined by luminometry of homogenized tissue harvested from the infection
site. A group of animals sham-treated with PBS was included as a control.
Leishmanicidal activity and percentage of macrophages, neutrophils, and cells
from lesions that were producing NO were determined by flow cytometry as
above.
Resident peritoneal macrophages were obtained from pregnant or control
hamsters by peritoneal lavage with RPMI-EDTA medium. To quantify parasites replicating within macrophages, 106 resident peritoneal macrophages
were distributed in 24-well plates and incubated for 24 h at 37°C, 5% CO2.
Adherent macrophages were infected with 5 ⫻ 106 LUC-transfected L. panamensis promastigotes and cultured at 34°C, 5% CO2, for 2 h. After washing
away extracellular parasites with warm Dulbeco’s PBS, macrophages were
cultured for an additional 72 h at 34°C and 5% CO2 to allow Leishmania
replication. Infected host cells were carefully detached with the plastic plunger
of a tuberculin syringe in cold PBS, counted, adjusted to 100,000 cells, and
lysed in 5⫻ LUC lysis buffer. The number of intracellular parasites was
determined by luminometry as described above.
Statistical analysis
NO determination
The fluorescent probe 4-amino-5-methylamino-2⬘,7⬘-difluorescein (DAF-FM;
Molecular Probes) diacetate was also used to detect the proportion of cells
producing NO production by flow cytometry. The specificity of NO determinations was established by using the iNOS-specific inhibitor L-N6-(1-iminoethyl) lysine (L-NIL; Cayman Chemical Co., Ann Arbor, MI, USA) dihydrochloride and the peroxynitrite scavenger manganese (III) tetrakis (4-benzoic
acid) porphyrin (Calbiochem, San Diego, CA, USA). The percentage of NOproducing cells was determined in peripheral blood neutrophils, resident
peritoneal macrophages, and cells derived from the skin at the site of infection
(7 days p.i.). Cells from skin were obtained by mechanical dissociation using
a Medimachine威 apparatus (BD Biosciences) following the instructions of the
manufacturer. In brief, cells (500,000 cells in 100 ␮L PBS) were incubated
with 0.5 ␮M DAF-FM during 30 min at room temperature in dark. The cells
were washed with PBS and incubated for 15 min to complete de-esterification
of the intracellular diacetates. Fluorescence intensity and percentage of gated
cells expressing NO were determined in the FL1 channel (515 nm). DAF-FMlabeled cells from uninfected animals were used to define the threshold for flow
cytometry analysis. Total NO production was evaluated in culture supernatants
of 2.5 ⫻ 106 peritoneal macrophages stimulated with antigen and 1000 nM
estrogen (48 h in 250 ␮l final volume) using the Griess reaction (Cayman
Chemical Co.). The specificity of iNOS-derived NO production in the supernatants was determined by the addition of 1 mM L-NIL. A TaqMan assay was
used to identify iNOS induced by estrogen. Briefly, Universal Master Mix
(Applied Biosystems, Foster City, CA, USA) was mixed with 150 ng reversetranscribed RNA (SuperScript II, Invitrogen, Carlsbad, CA, USA), 700 nM
each primer, and 100 nM TaqMan probe (forward 5⬘-tga gcc act gag ttc tcc taa
gg-3⬘; reverse 5⬘-tcc tat ttc aac tcc aag atg ttc tg-3⬘; probe 5⬘-6FAM-cgt gga cac
ttc ctt tgt ctg tgc tcc-TAMRA-3⬘) in a final volume of 25 ␮l. The reference gene
␥-actin was used as normalizer and detected in a validated multiplex assay
using a VIC-labeled TaqMan probe. The fold increase in iNOS expression was
calculated using nonstimulated macrophages as calibrator and the ⌬ comparative threshold method.
In vitro assays using estrogen or progesterone
To evaluate the influence of estrogen or progesterone on NO production,
resident peritoneal macrophages were obtained by peritoneal lavage from
uninfected hamsters or from hamsters at 7 days p.i. with L. panamensis. For
measurement of hormone-induced NO, we used macrophages but not neutrophils, as the latter could only be recovered in limited numbers from peripheral
blood. Cells were cultured for 5 min at 37°C, 5% CO2, 95% air, with different
concentrations of 17-␤ estradiol (10 –1000 nM) or the progesterone metabolite,
5␣-pregnane-3␣-ol-20 (pregnane; 1–100 ␮M, Sigma Chemical Co., St. Louis,
MO, USA). In some experiments, the estrogen receptor inhibitor tamoxifen (1
␮M) was used to antagonize the action of 17-␤-estradiol as described [24]. The
estrogen and tamoxifen were dissolved in absolute ethanol, and dilutions made
in RPMI, 5% FCS, 2 mM glutamine, 100 units/mL penicillin, and 100 ␮g/mL
streptomycin. After treatments, cells were cocultured with L. panamensis at
34°C, and parasite survival and proportion of cells producing NO were determined by flow cytometry. iNOS expression was quantitated by real-time PCR
as described above.
Multiple comparisons ANOVA with post-hoc Duncan’s test or Kruskal-Wallis
test with Dunn’s procedure were used at a 95% confidence interval using SPSS
standard program for Windows. Single comparisons were performed using
GraphPad InStat software, Version 3.06, as described in each figure.
RESULTS
Pregnancy enhances the control of early
cutaneous infection with L. panamensis
Female hamsters infected with L. panamensis during pregnancy developed significantly smaller lesions than nonpregnant controls during the early phase of infection (Fig. 1A).
This phase included the second half of pregnancy, and the
difference in lesion size was evident at the time of parturition
(15 days p.i.; P⫽0.040) and at the completion of lactation (30
days p.i.; P⫽0.038). In animals infected during pregnancy
(n⫽6), the draining lymph node harbored fewer parasites than
in nonpregnant females (n⫽4; P⫽0.02; Mann-Whitney U-test;
data not shown). In two different experiments, the number of
parasites was determined in lesion tissue using a LUC-transfected L. panamensis strain. We found that pregnant animals
had a 2.5-fold lower number of amastigotes in the infection site
than control animals (P⫽0.0098; Fig. 1B).
Pregnancy modulates the cellular immune
response to early L. panamensis infection
As it is generally accepted that cellular immune function is
altered during pregnancy, we first examined leukocyte populations in L. panamensis-infected pregnant and nonpregnant
hamsters. Pregnant, infected hamsters had a lower percentage
of peripheral blood lymphocytes than controls (P⫽0.01); however, there was a tendency toward an increased proportion of
CD4⫹ T cells in pregnant compared with nonpregnant, infected females (P⫽0.07; Table 1). No differences in the
proportion of B lymphocytes, mononuclear cells, or granulocytes were found among experimental groups (Table 1).
To identify the potential mechanisms for the enhanced resistance to early cutaneous infection during pregnancy, we first
examined the in situ cytokine response at the site of infection.
In general, there was greater expression of cytokine mRNAs in
the cutaneous lesion than in the draining lymph node, and
cytokine expression was equivalent to or reduced in the preg-
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis
1415
Fig. 1. Pregnancy enhances the control of
early cutaneous infection with L. panamensis. (A) Lesion size. Pregnant female
(shaded bars) or nonpregnant female (open
bars) hamsters were infected in the snout
with 104 L. panamensis promastigotes, and
the lesion size (mean⫾SD) was determined
after 15 days p.i. (parturition) and 30 days
p.i. (end of lactation). Individuals infected
during pregnancy (n⫽9) had smaller lesions
than controls (n⫽12) at both time-points
(P⫽0.04; unpaired t-test, normality tested
by Kolmogorov and Smirnov). (B) Parasite
burden. The parasite burden in the snout
from pregnant (n⫽7) and nonpregnant female hamsters (n⫽11), infected as described above, was determined by luminometry at 7 days p.i. Data are combined from two independent experiments and are
presented as number of amastigotes per lesion (mean⫾SE; P⫽0.0098; unpaired t-test).
nant animals (Table 2). Transcripts for the proinflammatory
and macrophage-activating cytokines IL-12p40, IFN-␥, and
TNF-␣ were reduced at the lesion site (IL-12, TNF-␣) or
draining lymph nodes (IFN-␥) in pregnant compared with
nonpregnant animals (P⬍0.05). The expression of the antiinflammatory, macrophage-inactivating cytokine TGF-␤ was
also consistently low in the lesion and draining lymph node in
pregnant compared with nonpregnant animals (Table 2). Thus,
there was a generalized pregnancy-associated reduction in the
early expression of cytokine mRNA in response to L. panamensis infection.
Pregnancy enhances the generation of NO and
leishmanicidal capacity of macrophages and
neutrophils exposed to L. panamensis
Despite the pregnancy-related reduction in cytokine expression at the site of infection, the consistent clinical and parasitological control of infection and low parasite burden of
pregnant hamsters was associated with high in situ expression
of iNOS (NOS2). Densitometry analysis of RT-PCR products
showed that iNOS mRNA expression levels in pregnant animals were 3.5- and 1.4-fold higher in infected skin (P⫽0.07)
and draining lymph nodes (P⫽0.02), respectively, than those
of infected, nonpregnant controls (Table 2). These observations
prompted us to evaluate the effect of pregnancy on leishmanicidal capacity and NO production of macrophages and neutro-
TABLE 1. Phenotype of Peripheral Blood Cells in Pregnant
Hamsters Infected with L. panamensis (7 Days p.i.) Compared with
Infected, Nonpregnant Controls
% Cells (mean ⫾
Lymphocytes
T CD4⫹ lymphocytes
B lymphocytes
Mononuclear cells
Granulocytes
a
SD)
Controls
(n ⫽ 7)
Pregnant
(n ⫽ 9)
P value
42 ⫾ 5
16 ⫾ 2
11 ⫾ 3
3⫾2
51 ⫾ 9
35 ⫾ 6
26 ⫾ 9
10 ⫾ 3
3⫾1
52 ⫾ 7
0.01b
0.07
0.5
0.5
0.8
a
Percentage of positive cells determined by flow cytometry. b Statistically
significant between control and pregnant groups by Kruskal Wallis test; 95%
confidence.
1416
Journal of Leukocyte Biology Volume 83, June 2008
phils exposed to L. panamensis. Resident peritoneal macrophages harvested from infected, pregnant (n⫽7) and infected,
nonpregnant control hamsters (n⫽6) showed similar phagocytic capacities determined 20 min after the uptake of CFSElabeled promastigotes (mean percentage of macrophages with
parasites⫾SD⫽3⫾10% and 3⫾6%, respectively). Accordingly, the number of parasites determined by luminometry in
adherent macrophages at 20 min–1 h p.i. was similar in pregnant hamsters and nonpregnant controls (mean number of
amastigotes⫾SE⫽309,773⫾54,062, n⫽11; 369,704⫾80,499,
n⫽14, respectively). However, after 72 h of culture, macrophages from infected, pregnant hamsters harbored 3.6-fold
fewer amastigotes than the corresponding infected, nonpregnant controls (P⫽0.0181; Fig. 2A). Microscopical examina-
TABLE 2. Cytokine and NO mRNA Expression in the Infection
Site and Cells from Draining Lymph Nodes of Pregnant Hamsters
Infected with L. panamensis (7 Days p.i.) Compared with
Nonpregnant, Infected Controlsa
mRNA/HPRTb
Lesion
IL-10
TGF-␤
IL-4
IL-12p40
IFN-␥
TNF-␣
iNOS
Lymph node
IL-10
TGF-␤
IL-4
IL-12p40
IFN-␥
TNF-␣
iNOS
Controls
Pregnant
(n ⫽ 4)
82 ⫾ 31
168 ⫾ 103
0.6 ⫾ 16
135 ⫾ 125
52 ⫾ 26
193 ⫾ 26
21 ⫾ 42
(n ⫽ 7)
231 ⫾ 172
16 ⫾ 10
0
0
13 ⫾ 8
0
15 ⫾ 10
(n ⫽ 4)
42 ⫾ 28
11 ⫾ 22
17 ⫾ 28
7 ⫾ 14
29 ⫾ 35
85 ⫾ 19
74 ⫾ 20
(n ⫽ 5)
50 ⫾ 12
4⫾9
0
0
4⫾5
0
21 ⫾ 12
Ratioc
P valued
1.9
15.2
0.03
19.2
1.79
2.2
0.28
0.14
0.01d
0.9
0.05d
0.2
0.03d
0.07
4.6
4.0
–
–
3.2
–
0.71
0.09
0.02d
–
–
0.02d
–
0.02d
a
Data are from a single experiment representative of two or three independent experiments. b RT-PCR results are expressed as a net value with reference to the HPRT gene, which is constitutively expressed. c Control/pregnant
ratio indicates the ratio of mRNA that is expressed by controls with reference
to pregnant animals. d Statistically significant difference between control and
pregnant groups (Mann-Whitney test).
http://www.jleukbio.org
Fig. 2. Pregnancy enhances the leishmanicidal capacity of macrophages and
neutrophils exposed to L. panamensis. (A)
Leishmanicidal capacity of macrophages.
Resident peritoneal macrophages isolated
from infected (7 days p.i.), pregnant and
nonpregnant hamsters were infected in vitro
with L. panamensis, and the parasite burden
was determined after 72 h by luminometry.
Shown is the number of amastigotes
(mean⫾SE) obtained in three independent
experiments using four to five hamsters per
group per experiment (P⫽0.018; unpaired
t-test). (B) Leishmanicidal capacity of neutrophils (N␾s). Sorted peripheral blood neutrophils were pooled and infected with CFSE-labeled promastigotes. The percentage of neutrophils harboring viable CFSE ⫹ L. panamensis was determined by
flow cytometry at 20 min–1 h p.i. Shown is the mean ⫾ SD percentage of neutrophils containing viable leishmania observed in one experiment that is representative
of two independent experiments. Neutrophils from pregnant hamsters (nine pooled samples) had greater leishmanicidal capacity than neutrophils from the
nonpregnant animals (seven pooled samples; P⫽0.018; unpaired t-test).
tions confirmed this observation; at 72 h of culture, macrophages from pregnant females (n⫽6) harbored 0.9 ⫾ 0.7
(mean⫾SD) amastigotes per macrophage, and macrophages of
control females (n⫽7) had 4.0 ⫾ 4.6 (mean⫾SD) amastigotes
per macrophage (P⫽0.037; Mann-Whitney test).
Leishmanicidal activity was also studied in neutrophils from
pregnant animals. The proportion of neutrophils that phagocytosed Leishmania was similar in pregnant and control neutrophils (pregnant⫽95⫾2.4, pooled sample, n⫽9; controls⫽90.3⫾9.2, pooled sample, n⫽7), as determined by the
proportion of sorted neutrophils containing Leishmania. However, the analysis of intracellular amastigote viability, using
CFSE-labeled parasites, showed that a lower percentage of
neutrophils from pregnant compared with nonpregnant hamsters contained viable Leishmania (P⫽0.0187; Fig. 2B).
L. panamensis-triggered NO production by macrophages or
neutrophils was determined in a fluorescence-based (DAF-FM)
flow cytometry assay. The specificity of the NO detection was
established by using the NO inhibitor L-NIL dihydrochloride
(Cayman Chemical Co.). No NO production by L. panamensis
promastigotes was detected using DAF-FM. Hamster peritoneal macrophages infected in vitro with L. panamensis promastigotes and cultured for 24 h with 10 –1000 ␮M L-NIL showed
a dose-dependent decrease in NO production (linear regression, P⫽0.01); NO production by these macrophages was
abolished at 1000 ␮M L-NIL (data not shown).
There was a trend toward a higher proportion of resident
peritoneal macrophages isolated from infected, pregnant
hamsters to produce NO compared with macrophages from
infected, nonpregnant animals (mean⫾SE of five experiments: pregnant, 44.8⫾2.8; control, 34.6⫾5.1; P⫽0.07).
Additionally, the percentage of NO-producing neutrophils
was 6.8-fold greater in L. panamensis-infected, pregnant
animals compared with infected, nonpregnant animals
(mean⫾SE of two different experiments: pregnant, 72.8⫾8.3;
control, 10.7⫾1.5; P⫽0.0001). Pregnancy also enhanced
the percentage of neutrophils producing NO in uninfected
animals (P⫽0.006). Collectively, these data indicate that
neutrophils from pregnant or infected animals show increased NO production over controls and that pregnancy
amplifies NO production induced by infection.
Estrogen and pregnane enhance macrophage NO
The efficient parasite control observed in pregnant animals
during the early stages of infection, which was associated with
high iNOS expression, suggested that reproductive hormones
could play a role in Leishmania containment. Therefore, we
determined the influence of the pregnancy-associated hormone
estrogen (17-␤ estradiol) and pregnane (a progesterone metabolite) on NO production by macrophages upon in vitro infection
with L. panamensis. Resident peritoneal macrophages isolated
from hamsters infected with L. panamensis (7 days p.i.) and
restimulated with Leishmania antigens showed a dose-dependent increase in production of NO after ex vivo exposure to
increasing doses of 17-␤ estradiol (P⫽0.01; Fig. 3A). Addition of 1 ␮M tamoxifen (which competitively blocks the estrogen receptor) to the culture medium before supplementation
with estrogen impaired the hormone-mediated increase in NO
production (P⫽0.056; Fig. 3A). Peritoneal macrophages from
L. panamensis-infected hamsters exposed to this hormone for
6 h showed a 35-fold increase in iNOS expression over nonstimulated macrophages (P⫽0.01; Fig. 3B). Tamoxifen inhibited iNOS expression induced by estrogen treatment, suggesting iNOS expression by a classic, receptor-mediated pathway
(P⫽0.01; Fig. 3B).
In addition, peritoneal macrophages from infected animals
stimulated for 48 h with L. panamensis antigen and estrogen
released more total NO to culture supernatants compared with
macrophages cultured with antigen alone (P⫽0.05). Addition
of the iNOS inhibitor L-NIL to antigen/estrogen-stimulated
macrophages blocked the induction of NO production
(P⫽0.02; Fig. 3C).
Naı̈ve macrophages also showed increased iNOS expression
and NO production upon estrogen treatment. The quantification of iNOS using a TaqMan assay showed that peritoneal
macrophages from uninfected hamsters increased iNOS expression fivefold after 16 h exposure to 1000 nM estrogen
(nontreated⫽onefold⫾0.45; estrogen⫽5.3-fold⫾1.86; P⫽0.018;
unpaired t-test). On the other hand, flow cytometry evaluations
showed that the percentage of macrophages producing NO
increased sixfold after estrogen treatment (nontreated⫽
3.4%⫾1.1; estrogen-treated⫽20.7%⫾12).
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis
1417
Fig. 3. Estrogen and progesterone modulate NO production and iNOS expression
in resident peritoneal macrophages from
hamsters infected with L. panamensis.
Resident peritoneal macrophages were
isolated from hamsters infected with L.
panamensis and exposed in vitro for 5 min
to estrogen (17-␤ estradiol; 0 –2000 nM),
with or without tamoxifen (1 ␮M) or pregnane (5␣-pregnant-3␣-ol-20; 1–100 ␮M).
The estrogen receptor inhibitor tamoxifen
was included 30 min before estrogen
treatment and L. panamensis infection.
(A) Effect of estrogen (E2) on the proportion of NO-producing cells determined by
flow cytometry. Estrogen was added to the
macrophage cultures that were stimulated
with L. panamensis promastigotes. The
data shown (mean⫾SD) are from a single
experiment representative of two independent experiments with four hamsters each;
the threshold in the experiment was established using uninfected hamsters
(Uninf). The frequency of NO-producing
cells increased with estrogen treatment
(P⫽0.01) and decreased by blocking the
estrogen receptor with tamoxifen (P⫽0.056;
Kruskal-Wallis test; nonparametric ANOVA).
(B) Estrogen-induced iNOS expression
(real-time PCR). iNOS mRNA expression
in peritoneal macrophages from hamsters
infected with L. panamensis 6 h after treatment with 1000 nM estrogen or estrogen ⫹ 1 ␮M tamoxifen (TX). Data of four replicates from pooled samples
of five animals are shown as fold increase with respect to cells cultured with medium alone. Estrogen treatment induced iNOS mRNA expression (P⫽0.01)
and was blocked with tamoxifen (P⫽0.01; unpaired t-test). (C) Effect of estrogen on iNOS-derived NO production. Resident peritoneal macrophages
(2.5⫻106) isolated from L. panamensis-infected female hamsters (n⫽5) were stimulated for 48 h with L. panamensis (L.p.) antigen (100,000 frozen-thawed
L. panamensis promastigotes) in the presence or absence of estrogen (1000 nM), with or without iNOS inhibitor L-NIL (1 mM). NO (mean⫾SD) released in
the supernatants after 48 h of in vitro culture, determined by Griess reaction, showed that estrogen treatment induced NO production (P⫽0.05), which was
blocked with the addition of tamoxifen (P⫽0.02; unpaired t-test). (D) Effect of progesterone on NO production. Resident peritoneal macrophages from
hamsters infected with L. panamensis were exposed in vitro to 1–100 ␮M pregnane, and following 1 h of in vitro coculture with L. panamensis, the
NO-producing cells were identified by reaction with DAF-FM diacetate and flow cytometry. The frequency of NO-producing cells was increased with
pregnane treatment (P⫽0.001; Mann-Whitney test).
Treatment of peritoneal macrophages with concentrations of
pregnane ranging from 1 to 100 ␮M significantly increased the
percent of NO-producing cells (sixfold) compared with cells
from infected or uninfected animals sham-treated with medium
alone (P⫽0.001; Fig. 3D). An increase in the percentage of
NO-positive cells was not observed at lower doses of pregnane
(1–500 nM).
In vivo administration of estrogen and pregnane
modulates host NO production and
parasite control
To determine the in vivo relevance of the previous ex vivo and
in vitro observations, different groups of hamsters were treated
with the aforementioned hormones. Estrogen or pregnane was
administered to hamsters every 48 h for 14 days, and the
animals were infected on Day 7 of hormone treatment with
LUC-transfected L. panamensis to determine NO-producing
resident peritoneal macrophages and peripheral blood neutrophils; parasite survival after in vitro infection of resident
peritoneal macrophages and peripheral blood neutrophils; and
parasite burden and NO producer cells at the infection site by
luminometry or flow cytometry, respectively.
1418
Journal of Leukocyte Biology Volume 83, June 2008
Seven days after infection of hamsters that had been treated
with estrogen or pregnane, we found that NO production by
resident peritoneal macrophages was greater in the hormonetreated compared with untreated animals. Resident macrophages collected from infected hamsters treated with estrogen
produced eightfold more NO than those treated with PBS
(P⫽0.001; Table 3). Also, there was a tendency of macrophages from pregnane-treated hamsters to secrete higher levels
of NO (threefold; not significant) than controls. Macrophages
from estrogen-treated hamsters demonstrated significantly
greater leishmanicidal capacity than controls; this enhancement in Leishmania killing ability could not be detected in
macrophages derived from animals treated with pregnane (Table 3). A significant inverse correlation was found in the
percentage of estrogen-induced, NO-producing cells and parasite survival in peritoneal macrophages (Spearman’s correlation; P⫽0.041).
In the hamsters treated with estrogen or pregnane, we also
found that NO production by peripheral blood neutrophils was
three- to fourfold higher than in neutrophils from infected,
control animals that had been sham-treated with PBS (Table 3).
In addition, peripheral blood neutrophils from infected animals
http://www.jleukbio.org
TABLE 3. Percentage of Cells Producing NO and Percentage of
Viable Parasites in Peritoneal Macrophages or Peripheral Blood
Neutrophils from Hamsters Treated with Estrogen (17-␤-Estradiol)
or Pregnane (5␣-Pregnant-3␣-ol-20)a
NOb
Group
Macrophage
Control (PBS)
Estrogen
Pregnane
Neutrophil
Control (PBS)
Estrogen
Pregnane
Mean ⫾
(%)
Viable parasitesc
P
valued
Mean ⫾
(%)
1⫾1
8⫾3
3⫾2
–
0.001e
NSe
69 ⫾ 4
50 ⫾ 6
72 ⫾ 2
–
0.001f
NSf
9⫾2
29 ⫾ 14
36 ⫾ 21
–
0.002
0.002
62 ⫾ 25
43 ⫾ 25
64 ⫾ 15
–
0.03f
NSf
SD
SD
P
valued
a
Data are representative of two experiments using four animals per group.
NO production evaluated using flow cytometry and DAF-FM. c Viable
CFSE⫹ parasites measured after the in vitro infection of cells obtained from
female hamsters at Day 14 of treatment (7 days p.i. with L. panamensis).
d
Comparison made between hormone-treated and PBS-treated groups. e Kruskal-Wallis test with post-hoc Dunn’s correction for multiple comparisons.
f
ANOVA; Duncan’s post-hoc test. NS, Not significant.
b
that had been treated with estrogen showed significantly
greater parasite killing following ex vivo infection than the
animals that did not receive estrogen (P⫽0.03; Table 3).
However, no differences were found in the leishmanicidal
capacity of neutrophils harvested from infected hamsters
treated with pregnane and those collected from infected animals not treated with the hormone (Table 3).
At 7 days p.i., prior to the detection of a measurable cutaneous lesion, the parasite burden in the inoculation site was
significantly lower in estrogen-treated hamsters than in controls (Fig. 4A; P⫽0.036). A similar but not significant trend
was observed in animals treated with pregnane (Fig. 4A).
Consistent with the in vitro observations, flow cytometry analysis showed that NO levels in the infected skin of hamsters
treated with estrogen were higher than in the corresponding
controls (P⫽0.001; Fig. 4B). Also, a negative correlation between the proportion of NO-producer cells and parasite burden
was found in the dermis of hamsters treated with estrogen or
pregnane (Spearman’s correlation; P⫽0.05).
DISCUSSION
The present study is the first to explore the role of phagocytes
in the innate immune responses to experimental cutaneous
leishmaniasis during pregnancy. The clinical and parasitological results clearly indicated that during the early stages of
infection, pregnant female hamsters were capable of controlling Leishmania more effectively than nonpregnant individuals.
This heightened control of Leishmania infection was paralleled
by enhanced innate immune responses, as demonstrated by
increased NO generation and parasite killing by macrophages
and neutrophils during the first week of infection. Pregnancy
acted additively with cutaneous infection to enhance iNOS
mRNA expression and NO production locally (inoculation site
and draining lymph node) and systemically (resident peritoneal
macrophages and peripheral blood neutrophils). The administration of the pregnancy-related hormones estrogen and progesterone to hamsters or to in vitro-cultured macrophages
recapitulated the increased leishmanicidal activity observed in
pregnancy.
The activation of innate immunity during pregnancy, as we
observed in this study in hamsters, has been described in
humans [12, 14, 15]. Furthermore, female hamsters that were
infected with L. infantum 24 h after parturition were also found
to have less evidence of visceral leishmaniasis and a reduced
visceral parasite burden [29]. The influence of elevated prolactin
was considered to be the cause of the increased resistance;
however, our data suggest that other residual, pregnancyrelated hormonal changes could have also contributed to the
enhanced resistance in this early challenge model. Our findings are distinct from what was reported for pregnant C57BL/6
mice infected with L. major, in which the latter were more
susceptible than nonpregnant females [7]. This apparent discrepancy could be in part a result of differences in the Leishmania strain used and/or host species between the studies. The
study of Krishnan et al. [7] focused principally on the devel-
Fig. 4. Hormone treatment modulates parasite burden and NO production at the site
of infection. Female hamsters (n⫽6 per
group) were treated with 2 ␮g 17-␤-estradiol (Estrogen) in PBS, 3 ␮g 5␣-pregnane3␣-ol-20 (Pregnane) in PBS, or PBS alone
every 48 h for 14 days. The animals were
infected with L. panamensis in the snout on
Day 7 of treatment. The number of animals
in each group is shown on the horizontal
axis. (A) Parasite burden at the site of inoculation. The parasite burden in the snout
was determined 7 days p.i. by luminometry.
The data (amastigote numbers) are shown as
a box plot (median plus first–third quartile)
with whiskers showing the smallest and
largest values. Significant differences were identified between PBS and estrogen treatment (P⫽0.036) but not between PBS and pregnane (Kruskal-Wallis test and
Dunn procedure). (B) NO-producing cells at the site of inoculation. NO production by cells in the infected snout tissue was evaluated by flow cytometry of
DAF-FM-labeled cells. Significant differences were identified between PBS and pregnane treatment (P⫽0.001) and PBS and estrogen (P⫽0.026; Kruskal-Wallis
test; nonparametric ANOVA).
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis
1419
opment of the adaptive, T cell-mediated immune response in a
chronic infection model rather than in the early events of
innate immunity as studied here. Nevertheless, our studies do
not exclude the possibility that females infected during pregnancy could ultimately develop a Th2 response that worsens
chronic infections.
Parasite internalization by resident peritoneal macrophages
was found to be unaltered during pregnancy, but macrophages
from pregnant, infected hamsters had enhanced capacity to
restrict Leishmania replication at 72 h p.i. The increased
leismanicidal activity of hamster macrophages and neutrophils
during pregnancy was accompanied by a general decrease in
expression of cytokines (IFN-␥, IL-12, and TNF-␣) typically
associated with classical macrophage microbicidal activity.
This cytokine down-regulation concurs with the classic paradigm of suppression of T cell responses (without a Th2 cytokine
bias) during human pregnancy [1–3, 13–15] but differs from a
previous report of an enhanced Th2 (IL-4) response in pregnant
mice infected with L. major [7]. The relatively greater leishmanicidal activity of macrophages from pregnant females, despite the reduced expression of classical macrophage activators, may be related to the reduced expression of TGF-␤ and to
a lesser extent, IL-10, cytokines known to suppress macrophage effector function and promote Leishmania infection [41].
Alternatively, macrophages of pregnant females might have
enhanced responsiveness to IFN-␥ through up-regulation of
expression of IFN-␥ receptors, as has been described in myometrial macrophages of pregnant mice [42].
Our results indicate that during the first days of infection,
not only macrophages but also neutrophils from pregnant females generated more NO and killed Leishmania more effectively than those from nonpregnant animals. The leishmanicidal capacity of neutrophils has also been demonstrated in
human cells infected ex vivo with L. major [43] in resistant
mouse strains [44] and in hamsters infected with L. panamensis
(unpublished), suggesting that they constitute an early, primary
barrier to Leishmania infection. However, other reports have
suggested that neutrophils could also act as a “vector” for
intracellular Leishmania via a parasite-induced delay in apoptosis [16, 43, 45] or through the phagocytosis of apoptotic,
amastigote-laden neutrophils by human macrophages. Our data
indicate that during pregnancy, the neutrophils were highly
activated and had enhanced parasite killing, so were probably
not acting as permissive Leishmania host cells.
Hamsters, like humans, express iNOS at levels substantially
lower than mice [36, 46]. We demonstrated previously that in
L. donovani-infected hamsters and in IFN-␥/LPS-stimulated
hamster macrophages, iNOS mRNA expression and NO production could not be detected by Northern blot and Greiss
reaction, respectively [36, 46]. The data presented here are not
incongruent with these previous findings for several reasons.
First, NO-producing macrophages and neutrophils were detected in this hamster model of cutaneous L. panamensis infection using a much more sensitive, fluorescence-based flow
cytometry assay, and similarly low levels of NO production and
iNOS were measured by Griess reaction and real-time PCR.
Second, parasite-specific differences in the induction of iNOS
1420
Journal of Leukocyte Biology Volume 83, June 2008
expression are evident; the relatively controlled, cutaneous L.
panamensis infection is accompanied by iNOS expression,
whereas the progressive, fatal L. donovani infection actually is
coincident with suppressed iNOS expression (our unpublished
observations). That the generation of reactive nitrogen species
is stimulus-specific is further supported by the demonstration
of iNOS expression and NO production in hamsters immunized
against L. donovani [47] and in hamsters infected with Entamoeba histolytica [48].
This work adds support to a growing number of reports, in
experimental animals and humans, that estrogen is important
for iNOS expression and NO production by different cell types,
including monocytes and macrophages [24, 26, 49]. The ex
vivo and in vitro studies presented here identify a direct effect
of estrogen on iNOS in macrophages, which was mediated
through the estrogen receptor (our data and refs. [25, 26]).
We confirmed that the estrogen-enhanced NO production
and iNOS expression found in cultured phagocytes were also
evident in vivo following administration of estrogen to nonpregnant hamsters. Most notably, the estrogen-enhanced phagocyte
leishmanicidal activity was associated with improved control of
the early phase of cutaneous infection. In addition to the direct
effect on macrophages, at the site of infection in vivo, the
estrogen effect might be mediated by T cell-dependent signals
[50, 51].
Physiological studies have also associated the increased
progesterone levels in pregnancy with the biosynthesis of NO
[27, 28, 40]. The administration of pregnane to hamsters increased the number of NO-producing cells at the site of cutaneous infection and by ex vivo-cultured macrophages and
neutrophils. However, there was no pregnane-mediated increase in parasite killing in the lesion or by ex vivo-cultured
macrophages or neutrophils. This suggests that a hormonemediated increase in NO production is not sufficient in and of
itself to affect parasite killing and that there must be other
estrogen-induced effector mechanisms that act additively or
synergistically with the generation of NO.
This experimental study demonstrates that the innate immune response, characterized by phagocyte NO production and
control of L. panamensis infection, is enhanced during pregnancy. Although ex vivo studies demonstrated that macrophages and neutrophils could be a source of the increased NO
production, and both are found at the site of infection [32],
further studies are needed to define more clearly the relative
contribution of these cells to the pregnancy-enhanced NO
production and parasite killing in vivo. To our knowledge, this
is the first study in which a clear association between estrogeninduced iNOS expression/NO production and Leishmania killing has been found. Significantly, this pregnancy- and hormone-enhanced innate host defense was demonstrated in an
animal model whose macrophages show relatively low iNOS
expression and NO production in response to inflammatory
stimuli, a feature similar to humans but distinct from the
murine rodents. These results underscore the importance of
hormones in the regulation of the immune response and support the concept that physiological changes that are associated
with varying levels of circulating hormones can impact the
http://www.jleukbio.org
balance between host and pathogens such as Leishmania.
Additional studies will need to explore the consequences of
pregnancy on the evolution of the adaptive immune response
and chronic disease. It will also be important to understand
how infection before or during pregnancy in humans could
affect the natural history of disease in the mother and the child.
The knowledge of mechanisms involved in the estrogen-induced production of NO is of potential interest for the control
of intracellular pathogens.
ACKNOWLEDGMENTS
This work was supported by the National Program of Science
and Technology in Health (Colombia; COLCIENCIAS),
Projects 22290412603 and 22290414328, and the National
Institutes of Health (Bethesda, MD, USA), grant AI 061624 to
P. C. M. We thank Dr. Mark Ouellete (University of Laval,
Canada) and Dr. John Walker (CIDEIM) for providing the
plasmid and transfection of the Leishmania panamensis strain
used in this study. We specially thank Osibar Jamauca for the
hamster husbandry. The laboratory assistance of Carlos A.
Hernandez and Hector Henao and the statistical support of
Mauricio Perez are gratefully acknowledged.
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